SUMMARY Identification of the parasite genes that influence transmission, virulence and host specificity is of central interest for understanding host/parasite interactions. The central aim of this proposal is to identify the genetic basis of two such traits in Schistosoma mansoni, a parasitic fluke that infects 67 million people in South America, Middle-East and Africa. We will focus on the larval stage of the parasite lifecycle in aquatic snails. Following penetration of the snail host, schistosome larvae reproduce clonally, castrating their snail host, and hundreds to tens of thousands of motile cercariae larvae are released into the water where they infect humans or rodents. Two key transmission- related traits show high levels of heritable genetic variation among parasites. First, the timing of cercarial release from the snail varies among populations and overlaps with the water contact patterns of their vertebrate hosts. Most S. mansoni populations that primarily infect humans shed cercariae larvae in late morning, while parasite populations that primarily infect rodents shed cercariae in late afternoon or night. Late shedding has evolved at least three times in S. mansoni populations. Laboratory crosses demonstrate a simple genetic basis for this trait, but the genetic architecture of late shedding is different in the three populations where it has been documented. Second, the number of cercariae larvae shed from the intermediate aquatic snail host varies ?7-fold among individual schistosome genotypes. High shedding parasites have greater potential for transmission but also castrate and kill their intermediate snails more rapidly than low shedding parasites. Laboratory experiments demonstrate that this trait responds rapidly to selection and is highly heritable. Schistosome parasites are unusual among parasites of humans because the complete lifecycle can be easily maintained in the laboratory, so genetic crosses can be staged and thousands of progeny isolated, while a complete genome sequence and a growing molecular toolkit now allows genomic and functional characterization. In preliminary work, we conducted genetic crosses between diurnal and nocturnal shedding parasites from Oman and identified a region on chr.1 that determines cercarial shedding time (LOD=6.1). In Aim 1, we will exploit the growing schistosome molecular toolkit to fine map and functionally analyze the gene(s) that determine shedding time in the Omani cross, and we will conduct additional crosses to determine the genetic basis of this trait in other parasite populations where late shedding is observed. In Aim 2, we will use RNAseq to examine rhythms in expression of nocturnal and diurnal shedding parasites from Oman across the 24 hr light/dark cycle, to investigate the metabolic pathways underlying control of cercarial release. Finally, in Aim 3, we will analyze genetic crosses between parasites showing 7-fold differences in numbers of cercariae shed from infected snails, and then fine map and functionally analyze the genes involved. We have previously used genetic linkage mapping and functional analysis to identify the mutations underlying oxamniquine resistance in S. mansoni. We anticipate that this approach will be equally powerful for identifying the genes that underlie heritable parasite traits that are critical for transmission, host specificity and virulence in this important human helminth parasite. Our genetic approach also provides a novel way to probe the biology of schistosome sporocysts, a neglected stage in the schistosome lifecycle that is key to understanding transmission from the snail to the vertebrate host.